Supplementary Figures 1 - 8 from Reversal of Acquired Drug Resistance in <i>FLT3</i>-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

Abstract

<p>PDF file - 468K, S1. Sorafenib-resistant murine AML (Ba/F3-ITD-Res) and parental cells (Ba/F3-ITD) were treated with indicated concentrations of sorafenib for 72 hours. Cell apoptosis induction was analyzed by measuring the percentage of annexin V-positive cells using flow cytometry. S2. Ba/F3-D835G, Ba/F3-D835Y and Ba/F3-FLT3 wild type cells were treated with crenolanib for 72 hours and their EC50s for apoptosis induction were determined by measuring percentage of annexin V-positive cells using flow cytometry. S3. Isobologram and combination index (CI) analyses were performed on sorafenib resistant cells after 48 hours combination treatment with crenolanib and sorafenib . S4. Isobologram and combination index (CI) analyses were performed on sorafenib resistant cells Ba/F3-ITD+676/842 after 48 hours combination treatment with vary combination strategies of type I and type II tyrosine kinase inhibitors. S5. Ba/F3-FLT3 wild type cells were treated with vary combination strategies of type I and type II tyrosine kinase inhibitors for 48 hours and apoptosis induction were determined by measuring percentage of annexin V-positive cells using flow cytometry. Creno: crenolanib; sora: sorafenib; PKC: PKC412; MLN: MLN518. S6. Normal bone marrow cells were exposed to indicated agents for 48 hours and apoptosis induction was determined by measuring the percentage of Annexin V-positive cells using flow cytometry. S7. FLT3 wild type AML patient blasts were exposed to crenolanib and/or sorafenib at physiologically achievable concentrations (i.e. Crenolanib @ 2 microM and sorafenib @ 5 microM) for 48 hours and apoptosis induction was determined by measuring the percentage of Annexin V-positive cells by gating CD33/CD34 positive population using flow cytometry. * Specific apoptosis (%) was calculated as follow: 100 X (drug-induced apoptosis − spontaneous apoptosis)/(100 − spontaneous apoptosis). S8. Sorafenib-resistan cells Ba/F3-ITD+676/842 were cultured in normoxic (21% oxygen) or hypoxic (1% oxygen) condition in the presence/absence of mesenchymal stem cells (MSC) feeder for 6 hours and phosphorylation levels of FLT3 and its downstream signaling ERK and AKT were determined using Western blotting.</p>